Sine Oculis Homeobox Homolog 1 Expression Research Articles

Deletion of adipocyte Sine Oculis Homeobox Homolog 1 prevents lipolysis and attenuates skin fibrosis.

Dermal fibrosis is a cardinal feature of systemic sclerosis (SSc) for which there are limited treatment strategies. This is in part due to our fragmented understanding of how dermal white adipose tissue (DWAT) contributes to skin fibrosis. We identified elevated sine oculis homeobox homolog 1 (SIX1) expression in SSc skin samples from the GENISOS and PRESS cohorts, the expression of which correlated with adipose-associated genes and molecular pathways. SIX1 localization studies identified increased signals in the DWAT area in SSc and in experimental models of skin fibrosis. Global and adipocyte specific Six1 deletion abrogated end-stage fibrotic gene expression and dermal adipocyte shrinkage induced by SQ bleomycin treatment. Further studies revealed a link between elevated SIX1 and increased expression of SERPINE1 and its protein PAI-1 which are known pro-fibrotic mediators. However, SIX1 deletion did not appear to affect cellular trans differentiation. Taken together these results point at SIX1 as a potential target for dermal fibrosis in SSc.

FOXC2-induced circCASK aggravates colorectal cancer progression by upregulating SIX1 expression.

Colorectal cancer (CRC) ranks as the most common gastrointestinal solid carcinoma globally. Substantial evidence has established a pivotal role for circular RNAs (circRNAs) in CRC progression. In this study, differentially expressed circRNAs were analyzed based on a public dataset (GSE126094) and elevated expression of circCASK (hsa_circ_0001917) was validated in CRC. Moreover, increased circCASK was also confirmed in CRC patients. Functionally, circCASK knockdown led to a significant decrease in CRC cell growth and attenuated cell migration and invasion. Similarly, circCASK knockdown markedly attenuated tumor growth in vivo. Mechanistically, circCASK sponged miR-1271-5p and enhanced sine oculis homeobox homolog 1 (SIX1) expression. More importantly, both SIX1 overexpression and miR-1271-5p knockdown could reverse the cellular behavior inhibition induced by circCASK knockdown. Furthermore, SIX1 was most strongly and positively linked with Wnt/β-catenin signaling pathways, circCASK triggered Wnt/β-catenin signaling through the miR-1271-5p/SIX1 axis, and FOXC2 transcriptionally induced circCASK expression. In conclusion, circCASK induced by FOXC2 accelerated CRC progression through the miR-1271-5p/SIX1 axis, thus providing an interesting insight into CRC tumorigenesis.

The SIX1/LDHA Axis Promotes Lactate Accumulation and Leads to NK Cell Dysfunction in Pancreatic Cancer.

Pancreatic cancer (PC) is a malignant cancer with poor prognosis and high mortality rate. Sine oculis homeobox homolog 1 (SIX1) participates in the development of many cancers. However, the function of SIX1 in PC is not fully understood. SIX1 expression was determined using immunohistochemistry in PC tissues and cell lines. Glucose consumption, lactate production, and ATP assays were used to detect the function of SIX1. PC cells and NK cells were cocultured to study the effect of SIX1 overexpression in PC cells on NK cell function. Chromatin immunoprecipitation (ChIP) assays were used to study the relationship between SIX1 and lactate dehydrogenase A (LDHA). A series of in vitro and in vivo assays were further applied to elucidate the important role of the SIX1/LDHA axis in metabolism and NK cell dysfunction in PC. SIX1 was significantly upregulated in PC tissue; SIX1 overexpression promoted the glycolysis capacity of PANC-1 and CFPAC-1 cells and resulted in NK cell dysfunction after the NK cells had been cultured with PC cells. LDHA inhibitor partially restored the promotion of PC caused by SIX1 overexpression. According to ChIP assays, SIX1 directly binds to the LDHA promoter region. Moreover, LDHA inhibitor and lactate transporter blocker treatment promoted the function of NK cells cocultured with PC cells. In vivo experiments yielded the same results. The SIX1/LDHA axis promotes lactate accumulation and leads to NK cell dysfunction in PC.

SIX1: A Prognostic Biomarker in Uterine Corpus Endometrial Carcinoma.

Uterine Corpus Endometrial Carcinoma (UCEC) is a common malignancy of the female genital tract. The sine oculis homeobox homolog 1 (SIX1) protein has been documented to be important for tumor progression. However, little is known about the relationship between SIX1 and the pathogenesis of UCEC. This study aimed to assess the prognostic value of biomarker SIX1 in UCEC by analyzing clinical traits, immune infiltration, and gene set enrichment analysis. The Wilcoxon signed-rank test and logistic regression were used to analyze the relationship between clinicopathological characteristics and SIX1. The Kaplan-Meier method was used to assess the relationship between clinicopathological characteristics and prognosis verified by immunohistochemistry (IHC). Then gene set enrichment analysis (GSEA) was performed to explore signaling pathways correlated with SIX1 expression in UCEC. Finally, the TIMER2 database was used to analyze the correlation between SIX1 and immune infiltration, and the effect of SIX1 expression on immune cells was calculated with the CIBERSORT algorithm. We found that the expression of SIX1 in UCEC was up-regulated and correlated with a poor prognosis. Analysis showed that the expression of SIX1 was related to various clinical features and was an independent prognostic factor of UCEC. Enrichment analysis showed that SIX1 promoted the occurrence and development of UCEC by regulating multiple signaling pathways. The results of immune infiltration analysis showed that SIX1 has a complex correlation with immune infiltration. Our findings indicate that SIX1 is a promising biomarker for predicting the prognosis of UCEC and is a potential therapeutic target.

Tanshinone IIA inhibits cell growth by suppressing SIX1‑induced aerobic glycolysis in non‑small cell lung cancer cells

Aerobic glycolysis plays a key role in cancer cell metabolism and contributes to tumorigenesis, including that of non-small cell lung cancer (NSCLC). Tanshinone IIA (Tan IIA), an active compound of Salvia miltiorrhiza, exhibits antitumor properties. Multiple mechanisms are involved in the antitumor action of Tan IIA in lung cancer, such as inhibiting cell growth, promoting cell apoptosis and influencing cellular metabolism. However, the effects of Tan IIA on NSCLC cells and its mechanisms of action remain unclear. The present study shows Tan IIA dose-dependently attenuated the growth of NSCLC cells and in vitro in a dose-dependent manner. Moreover, Tan IIA markedly decreased the ATP level, glucose uptake and lactate production in the NSCLC cells in vitro. Tan IIA also inhibited tumor growth in a xenograft model in vivo. Mechanically, Tan IIA treatment decreased sine oculis homeobox homolog 1 (SIX1) mRNA and protein levels, thus leading to the downregulation of pyruvate kinase isozyme M2, hexokinase 2 and lactate dehydrogenase A (LDHA) expression in A549 cells. SIX1 knockdown with small interfering-RNA inhibited glycolysis in NSCLC cells, suggesting that SIX1 plays a role in the antitumor effect of Tan IIA on NSCLC cells. More importantly, it was demonstrated that SIX1 expression was stimulated in patients with NSCLC and was positively correlated with the LDH serum level. Finally, SIX1 low expression levels predicted the poor prognosis of patients with NSCLC. In conclusion, the present study showed that Tan IIA functioned as an anti-glycolysis agent in NSCLC cells by downregulating SIX1 expression and inhibiting cell proliferation.

SIX1 Predicts Poor Prognosis and Facilitates the Progression of Non-small Lung Cancer via Activating the Notch Signaling Pathway.

Background: Many transcription factors involved in embryonic development and reactivated in tumors are considered potential prognostic biomarkers and novel therapeutic targets in various cancers. Sine oculis homeobox homolog 1 (SIX1), a developmentally restricted transcriptional regulator, plays a critical role during tumor initiation and development. However, the prognostic value and biological function of SIX1 in non-small cell lung cancer (NSCLC) remain unclear.Methods: Bioinformatic analyses were conducted to investigate the expression of SIX1 in cancer and adjacent normal tissues of NSCLC and further explore the correlations between SIX1 expression and clinical outcomes. Western blotting and RT-PCR analysis were performed to detect of SIX1 expression level in NSCLC cell lines and normal bronchial epithelial cell. EdU, CCK-8, clonal formation assay, wound healing and transwell assay were performed to explore the effects of gain- or loss-of-function of SIX1 on cellular proliferation, migration and invasion in vitro. Gene set enrichment analysis (GSEA) was used to identify the potential signaling pathways involved in SIX1 mediated biological function and the correlation was confirmed by western blotting and RT-PCR analysis. In vivo experiment was conducted to further validate the tumor-promoting effects of SIX1.Results: Bioinformatic analysis indicated that SIX1 was markedly upregulated in NSCLC tissues of and positively correlated with poor prognosis of patients with NSCLC. Ectopic expression of SIX1 facilitated proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) of NSCLC cells. On the contrary, knocking down SIX1 exhibited the opposite effects. Mechanistic studies suggested that SIX1 activated the Notch pathway to promote the malignant biological behaviors of NSCLC, which could be reversed by inhibiting the Notch signaling with γ-secretase inhibitor.Conclusions: SIX1 could facilitate multiple malignant biological behaviors by activating the Notch signaling pathway and function as a promising prognostic biomarker.

NIK-SIX1 signalling axis regulates high glucose-induced endothelial cell dysfunction and inflammation

Endothelial dysfunction and inflammation are the main manifestations of diabetes-associated atherosclerosis. This paper studied the roles of NF-κB-inducing kinase (NIK) and sine oculis homeobox homolog 1 (SIX1) in regulating high glucose-induced endothelial dysfunction and inflammation. The expression of NIK and SIX1 in human umbilical vein endothelial cells (HUVECs) was silenced by transfection with the specific shRNAs. HUVECs exposed to high glucose were considered as a cell model of endothelial dysfunction. Expression of NIK and SIX1 following transfection was measured by qRT-PCR and western blotting analysis. The proliferation, migration, and inflammation of HUVECs were evaluated by EdU staining, scratch test, ELISA, and western blotting. High glucose (30 mM) significantly decreased the proliferation and migration of HUVECs. High glucose-induced the expression of adhesion molecules VCAM-1 and ICAM-1. Moreover, high glucose increased the release of IL-1β, IL-6, TNF-α, and MCP-1. Transfection of cells with NIK shRNA significantly reversed the toxic effects of high glucose on HUVECs. Of contrast, SIX1 shRNA accelerated the effects of high glucose on HUVECs. NIK shRNA inhibited the accumulation of RelA, RelB, and p52. Meanwhile, NIK shRNA led to SIX1 downregulation which further induced the activation of the NF-κB pathway. NIK-SIX1 signalling axis was suggested to be critical in the regulation of high glucose-induced endothelial dysfunction and inflammation. SIX1 may function as an immunological gatekeeper to control the excessive inflammation mediated by NIK in diabetes-associated atherosclerosis.

MiR‑106b‑5p targeting SIX1 inhibits TGF‑β1‑induced pulmonary fibrosis and epithelial‑mesenchymaltransition in asthma through regulation of E2F1.

Asthma is an inflammatory disease of the airways, characterized by lung eosinophilia, mucus hypersecretion by goblet cells and airway hyper-responsiveness to inhaled allergens. The present study aimed to identify the function of microRNA (miR/miRNA)-106b-5p in TGF-β1-induced pulmonary fibrosis and epithelial-mesenchymal transition (EMT) via targeting sine oculis homeobox homolog 1 (SIX1) through regulation of E2F transcription factor 1 (E2F1) in asthma. Asthmatic mouse models were induced with ovalbumin. miRNA expression was evaluated using reverse transcription-quantitative PCR. Transfection experiments using bronchial epithelial cells were performed to determine the target genes. A luciferase reporter assay system was applied to identify the target gene of miR-106b-5p. The present study revealed downregulated miR-106b-5p expression and upregulated SIX1 expression in asthmatic mice and TGF-β1-induced BEAS-2B cells. Moreover, miR-106b-5p overexpression inhibited TGF-β1-induced fibrosis and EMT in BEAS-2B cells, while miR-106b-5p-knockdown produced the opposite effects. Subsequently, miR-106b-5p was found to regulate SIX1 through indirect regulation of E2F1. Additionally, E2F1- and SIX1-knockdown blocked TGF-β1-induced fibrosis and EMT in BEAS-2B cells. In addition, miR-106b-5p negatively regulated SIX1 via E2F1 in BEAS-2B cells. The present study demonstrated that the miR-106b-5p/E2F1/SIX1 signaling pathway may provide potential therapeutic targets for asthma.

Silencing SIX1 inhibits epithelial mesenchymal transition through regulating TGF-β/Smad2/3 signaling pathway in papillary thyroid carcinoma

ObjectiveTo investigate the sineoculis homeobox homolog 1 (SIX1) affect the epithelial mesenchymal transition (EMT) in papillary thyroid carcinoma (PTC) through regulating TGF-β/Smad2/3 signaling pathway. MethodsThe SIX1 expression in cytological specimens, tissues or PTC cell lines was detected by qRT-PCR, western blotting or immunohistochemistry. A series of vitro experiments including flow cytometry, CCK-8, wound-healing and Transwell were used to evaluate the biological characteristics in a PTC cell line (NPA cells), which were divided into Blank, Negative control (NC), SIX1, SIX1-siRNA, LY-364947 (TGF-β/Smad2/3 pathway inhibitor) and SIX1 + LY-364947 groups. TGF-β/Smad2/3 pathway and EMT related protein expression were measured by qRT-PCR and western blotting. ResultsSIX1 mRNA expression was increased in cytological specimens from PTC patients as compared with the non-toxic nodular goitre (NTG) patients. Moreover, compared with adjacent normal tissues, expressions of SIX1, N-cadherin and Vimentin were higher while E-cadherin was lower in PTC tissues; and SIX1 was positively correlated with N-cadherin and Vimentin but was negatively correlated with E-cadherin. Furthermore, the SIX1 expression was associated with histopathology, extrathyroidal extension (ETE), lymph node metastasis (LNM), pT stage, TNM stage, and distant metastasis. In addition, the expressions of TGFβ1, p-SMAD2/3, N-cadherin and Vimentin were downregulated in NPA cells after LY-364947 treatment with upregulated E-cadherin, decreased cell proliferation and metastasis, and enhanced cell apoptosis, which was reversed by SIX1 overexpression. ConclusionSilencing SIX1 can inhibit TGF-β/Smad2/3 pathway, thereby suppressing EMT in PTC, which may be a novel avenue for the treatment of PTC.

Evaluation of the expression level of Sine oculis homeobox homolog 1 in cervical cancer tissue in comparison with healthy adjacent tissue

Background and Aim: This study aimed to investigate the expression level of Sine oculis homeobox homolog 1(SIX1) in normal and cancerous cervical tissues. Methods: Real-time PCR was used to evaluate SIX1 expression level in fresh surgically obtained cervical biopsies. Also, cervical specimens were tested for HPV DNA detection by MY09/11, GP5+/6+ nested PCR method. Results: The expression level of SIX1 mRNA in Cervical cancer was markedly higher than in normal cervical tissue (p<0.0001). Moreover, HPV DNA was detected in 100% of cases and 21.05% of controls (P<0.0001). Conclusions: This finding indicates that increased SIX1 expression might be of relevance to the pathogenesis of cervical cancer. Perhaps, studies to find agents that specifically target SIX1, develop novel anti-cancer and anti-metastasis drugs. This idea has to be investigated in future clinical studies.

SIX1 cooperates with RUNX1 and SMAD4 in cell fate commitment of M\xfcllerian duct epithelium

During female mammal reproductive tract development, epithelial cells of the lower Müllerian duct are committed to become stratified squamous epithelium of the vagina and ectocervix, when the expression of ΔNp63 transcription factor is induced by mesenchymal cells. The absence of ΔNp63 expression leads to adenosis, the putative precursor of vaginal adenocarcinoma. Our previous studies with genetically engineered mouse models have established that fibroblast growth factor (FGF)/mitogen-activated protein kinase (MAPK), bone morphogenetic protein (BMP)/SMAD, and activin A/runt-related transcription factor 1 (RUNX1) signaling pathways are independently required for ΔNp63 expression in Müllerian duct epithelium (MDE). Here, we report that sine oculis homeobox homolog 1 (SIX1) plays a critical role in the activation of ΔNp63 locus in MDE as a downstream transcription factor of mesenchymal signals. In the developing mouse reproductive tract, SIX1 expression was restricted to MDE within the future cervix and vagina. SIX1 expression was totally absent in SMAD4 null MDE and was reduced in RUNX1 null and FGFR2 null MDE, indicating that SIX1 is under the control of vaginal mesenchymal factors: BMP4, activin A and FGF7/10. Furthermore, Six1, Runx1, and Smad4 gene-dose-dependently activated ΔNp63 expression in MDE within the vaginal fornix. Using a mouse model of diethylstilbestrol (DES)-associated vaginal adenosis, we found DES action through epithelial estrogen receptor α (ESR1) inhibits activation of ΔNp63 locus in MDE by transcriptionally repressing SIX1 and RUNX1 in the vaginal fornix.

Identification of a Small-Molecule Inhibitor That Disrupts the SIX1/EYA2 Complex, EMT, and Metastasis.

Metastasis is the major cause of mortality for patients with cancer, and dysregulation of developmental signaling pathways can significantly contribute to the metastatic process. The Sine oculis homeobox homolog 1 (SIX1)/eyes absent (EYA) transcriptional complex plays a critical role in the development of multiple organs and is typically downregulated after development is complete. In breast cancer, aberrant expression of SIX1 has been demonstrated to stimulate metastasis through activation of TGFβ signaling and subsequent induction of epithelial-mesenchymal transition (EMT). In addition, SIX1 can induce metastasis via non-cell autonomous means, including activation of GLI-signaling in neighboring tumor cells and activation of VEGFC-induced lymphangiogenesis. Thus, targeting SIX1 would be expected to inhibit metastasis while conferring limited side effects. However, transcription factors are notoriously difficult to target, and thus novel approaches to inhibit their action must be taken. Here we identified a novel small molecule compound, NCGC00378430 (abbreviated as 8430), that reduces the SIX1/EYA2 interaction. 8430 partially reversed transcriptional and metabolic profiles mediated by SIX1 overexpression and reversed SIX1-induced TGFβ signaling and EMT. 8430 was well tolerated when delivered to mice and significantly suppressed breast cancer-associated metastasis in vivo without significantly altering primary tumor growth. Thus, we have demonstrated for the first time that pharmacologic inhibition of the SIX1/EYA2 complex and associated phenotypes is sufficient to suppress breast cancer metastasis. SIGNIFICANCE: These findings identify and characterize a novel inhibitor of the SIX1/EYA2 complex that reverses EMT phenotypes suppressing breast cancer metastasis.

Increased SIX-1 expression promotes breast cancer metastasis by regulating lncATB-miR-200s-ZEB1 axis.

Patients with advanced breast cancer (BC) showed a higher incidence of regional and distant metastases. Sine oculis homeobox homolog 1 (SIX‐1) has been confirmed to be a key tumorigenic and metastatic regulator in BC progression. Yet, molecular mechanisms behind SIX‐1‐induced BC metastases remain largely unknown. Here we found that SIX‐1 was frequently up‐regulated in BC and correlated with poor outcomes when tested in human BC tissue microarray. Then, we manipulated the expression of SIX‐1 by via shRNA‐mediated knockdown and lentivirus‐mediated overexpression. Transwell assay in vitro and lung metastases model of nude mice in vivo showed that SIX‐1 promoted BC cell invasion and migration in vitro, and facilitated metastases in vivo. Mechanistically, SIX‐1 could promote the transcription of lncATB, which exerts critical pro‐metastatic role in BC by directly binding to the miR‐200 family, especially for miR‐200c, to induce EMT and promote metastases. In conclusion, SIX‐1 exerts its pro‐metastatic role in BC through lncATB/miR‐200s axis of EMT signalling pathway and could act as an important diagnostic marker as well as a significant therapeutic target for clinically advanced BC.

Down-regulation of MIAT suppresses osteosarcoma progression by acting as a ceRNA for miR-141-3p to regulate SIX1-mediated PI3K/AKT pathway.

Osteosarcoma (OS) is a frequent bone malignancy. Long non-coding RNA myocardial infarction associated transcript (MIAT) has been reported to be involved in the development of human cancers, including OS. However, the mechanism underlying MIAT in OS progression remains largely unclear. The expression levels of MIAT and sineoculis homeobox homolog 1 (SIX1) in OS tissues and cells were detected by quantitative real-time polymerase chain reaction and Western blot. Cell viability, apoptosis, migration and invasion of OS cells were determined by MTT, flow cytometry and trans-well assays, respectively. The target interaction among MIAT, miR-141-3p and SIX1 was analyzed by bioinformatics analysis and luciferase reporter assay. Phosphatidylinositide 3-kinases (PI3K)/protein kinase B (AKT) pathway was evaluated by Western blot. MIAT and SIX1 expression levels were enhanced in OS tissues and cells. Knockdown of MIAT or SIX1 repressed cell viability, migration and invasion but promoted apoptosis in OS cells. Moreover, overexpression of SIX1 reversed the inhibitive role of MIAT silence in OS progression. Furthermore, MIAT could increase SIX1 expression by competitively sponging miR-141-3p. Besides, inhibition of MIAT blocked PI3K/AKT pathway by decreasing SIX1 in OS cells. MIAT silence suppresses OS progression through inactivating PI3K/AKT signaling by sponging miR-141-3p to regulate SIX1, indicating a novel target for the treatment of OS.

MicroRNA-23a inhibits endometrial cancer cell development by targeting SIX1.

The present study focused on exploring the inhibitory mechanism of microRNA (miR)-23a in endometrial cancer. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was used to investigate miR-23a expression in endometrial tissues and endometrial cancer cells. A colony formation assay using crystal violet staining was performed to compare cell proliferation, while wound-healing and Transwell assays were performed to compare cell migration and invasion. Subsequently, bioinformatics and a luciferase reporter gene assay were used to investigate the effect of miR-23a on sine oculis homeobox homolog 1 (SIX1) expression, and the biological function of SIX1 was analyzed. Additionally, a nude mouse tumorigenicity assay was performed to test the inhibitory effect of miR-23a and Taxol® therapy in endometrial cancer. Finally, immunohistochemistry and RT-qPCR were used to explore the association between miR-23a and SIX1 expression in endometrial cancer tissues. miR-23a was underexpressed in endometrial cancer tissues compared with in para-carcinoma tissues, and the overexpression of miR-23a inhibited proliferation and invasion of endometrial cancer cells. Furthermore, SIX1 was demonstrated to be a downstream target of miR-23a, and miR-23a reduced SIX1 expression. Additionally, SIX1 inversely promoted cell proliferation, migration and invasion. In addition, the effects of reduced cell proliferation and increased cell invasion following miR-23a overexpression could be reversed by adding SIX1 to in vitro culture. Furthermore, the inhibitory effect of miR-23a and Taxol therapy, which reduced SIX1 expression in endometrial cancer, was demonstrated in vivo. Finally, a negative association between miR-23a and SIX1 expression was demonstrated in endometrial cancer tissues. The results of the present study revealed that miR-23a may inhibit endometrial cancer development by targeting SIX1.

MiR‑23b‑3p promotes the apoptosis and inhibits the proliferation and invasion of osteosarcoma cells by targeting SIX1.

Osteosarcoma (OS) is the most common primary malignant bone tumor and the third most common cancer that occurs during childhood and adolescence. Increasing evidence has suggested that microRNA (miR)‑23b‑3p has an important role in OS tumorigenesis; however, the underlying molecular mechanisms remain unknown. The aim of the present study was to investigate the expression levels of miR‑23b‑3p and sine oculis homeobox homolog 1 (SIX1) in OS tissues and cell lines (MG‑63, SaOS‑2 and U2OS), as well as to observe the effects of miR‑23b‑3p on U2OS cell viability, cell cycle, apoptosis and invasive ability. The results revealed that the expression levels of miR‑23b‑3p were significantly decreased in OS tissues and cell lines compared with tumor‑adjacent normal tissues and a non‑cancerous human fetal osteoblastic cell line (hFOB1.19). To investigate the underlying mechanisms of miR‑23b‑3p in OS tumorigenesis and progression, human U2OS cell lines over‑ or under expressing miR‑23b‑3p were established. The effects of miR‑23b‑3p on U2OS cell viability, cell cycle, apoptosis and invasion properties were determined by performing Cell Counting Kit‑8, flow cytometry and Transwell invasion assays. miR‑23b‑3p was revealed to suppress cell viability, proliferation and invasion, and to enhance the levels of cell apoptosis. Furthermore, SIX1 mRNA and protein expression levels in OS tissues and cell lines were significantly upregulated when compared with tumor‑adjacent normal tissues and hFOB 1.19 cells, which suggested that SIX1 expression levels may be inversely associated with miR‑23b‑3p levels in OS. Luciferase reporter system analysis demonstrated that miR‑23b‑3p binds to the SIX1 3'‑untranslated region. miR‑23b‑3p downregulation contributed to SIX1 upregulation, which facilitated the potentiation of cyclin D1 and vascular endothelial growth factor‑C expression levels, as well as the inhibition of caspase‑3 expression. Collectively, these results suggested that miR‑23b‑3p is downregulated and SIX1 is upregulated in OS cells, and that miR‑23b‑3p inhibition may suppress the proliferation and invasion of OS cells, and contribute to cell apoptosis via negative regulation of SIX1. miR‑23b‑3p/SIX1 may therefore represent a potential target for the treatment of OS.

The Sineoculis Homeobox Homolog 1 (SIX1) Gene Regulates Paclitaxel Resistance by Affecting Reactive Oxygen Species and Autophagy in Human Hepatocellular Carcinoma Cell Line HepG2.

BackgroundThe objective of this study was to explore the role of SIX1 in paclitaxel (TAX) resistance of HepG2 cells via reactive oxygen species (ROS) and autophagy pathway.Material/MethodsHepatoma cell line HepG2 was treated with SIX1 knockdown or/and TAX. Cell growth was detected by MTT assay and colony formation assay. Cell apoptosis was evaluated with flow cytometry. ROS levels were detected using flow cytometry (stained with DCFH2-DA). Western blot was conducted to detect the expression of SIX1 and autophagy-related proteins.ResultsTAX suppressed the proliferation of HepG2 cells in a time/dose-dependent manner, and upregulated the expression of SIX1. SIX1 siRNA increased TAX sensitivity of HepG2 cells and upregulated cell ROS levels. SIX1 siRNA combined with TAX treatment activated autophagy of HepG2 cells. N-acetyl-L-cysteine (NAC) partially attenuated SIX1 siRNA-induced ROS level increases, and autophagy inhibitor 3-MA notably enhanced SIX1 siRNA-induced cell apoptosis.ConclusionsKnockdown of SIX1 increased cell ROS levels and autophagy, promoted cell apoptosis, and enhanced TAX sensitivity of HepG2 cells.

SIX1 is overexpressed in endometrial carcinoma and promotes the malignant behavior of cancer cells through ERK and AKT signaling.

The sineoculis homeobox homolog 1 (SIX1) protein has been found to be important for cancer progression. However, its biological role in human endometrial carcinomas remains unexplored. The potential mechanism of SIX1-induced cancer progression remains unclear. In the present study, SIX1 protein expression was examined in 84 cases of endometrial carcinoma tissues using immunohistochemisty, and SIX1 was found to be overexpressed in 51.1% (43/84) of cervical cancer cells. Small interfering RNA (siRNA) knockdown of SIX1 was also performed in Ishikawa cells with high endogenous SIX1 expression, and SIX1 was overexpressed in the HEC1B cell line with low endogenous expression. SIX1 overexpression promoted cell growth rate and colony formation ability, whereas SIX1 depletion inhibited cell growth and colony formation. Further analysis showed that SIX1 knockdown downregulated, and SIX1 overexpression upregulated, cyclin D1, cyclin E, phosphorylated (p-)extracellular signal-regulated kinase (ERK), and p-protein kinase B (AKT) expression. The ERK inhibitor, U0126, and AKT inhibitor treatments blocked the effect of SIX1 on proliferation. In conclusion, the present study found that SIX1 overexpression promotes cancer cell growth in endometrial carcinoma, possibly through ERK- and AKT-mediated pathways.

SIX1 coordinates with TGFβ signals to induce epithelial-mesenchymal transition in cervical cancer.

Epithelial-mesenchymal transition (EMT) plays a critical role in promoting tumor invasion and metastasis. However, the key cofactors that modulate the signal transduction to induce EMT have note been fully explored to date. The present study reports that sine oculis homeobox homolog 1 (SIX1) is able to promote EMT of cervical cancer by coordinating with transforming growth factor (TGF)β-SMAD signals. The expression of SIX1 was negatively correlated with the expression of the epithelial marker E-cadherin in two independent groups of cervical cancer specimens. SIX1 could promote the transition of mesenchymal phenotype in the presence of active TGFβ signals in vitro and in vivo. TGFβ-SMAD signals were required for the SIX1-mediated promotion of EMT and metastatic capacity of cervical cancer cells. Together, SIX1 and TGFβ cooperated to induce more remarkable changes in the transition of phenotype than each of them alone, and coordinated to promote cell motility and tumor metastasis in cervical cancer. These results suggest that the coordination of SIX1 and TGFβ signals may be crucial in the EMT program, and that SIX1/TGFβ may be considered a valuable marker for evaluating the metastatic potential of cervical cancer cells, or a therapeutic target in the treatment of cervical cancer.

SIX1 promotes tumor lymphangiogenesis by coordinating TGFβ signals that increase expression of VEGF-C.

Lymphatic vessels are one of the major routes for the dissemination of cancer cells. Malignant tumors release growth factors such as VEGF-C to induce lymphangiogenesis, thereby promoting lymph node metastasis. Here, we report that sine oculis homeobox homolog 1 (SIX1), expressed in tumor cells, can promote tumor lymphangiogenesis and lymph node metastasis by coordinating with TGFβ to increase the expression of VEGF-C. Lymphangiogenesis and lymph node metastasis in cervical cancer were closely correlated with higher expression of SIX1 in tumor cells. By enhancing VEGF-C expression in tumor cells, SIX1 could augment the promoting effect of tumor cells on the migration and tube formation of lymphatic endothelial cells (LEC) in vitro and lymphangiogenesis in vivo. SIX1 enhanced TGFβ-induced activation of SMAD2/3 and coordinated with the SMAD pathway to modulate VEGF-C expression. Together, SIX1 and TGFβ induced much higher expression of VEGF-C in tumor cells than each of them alone. Despite its effect in promoting VEGF-C expression, TGFβ could inhibit lymphangiogenesis by directly inhibiting tube formation by LECs. However, the increased production of VEGF-C not only directly promoted migration and tube formation of LECs but also thwarted the inhibitory effect of TGFβ on LECs. That is, tumor cells that expressed high levels of SIX1 could promote lymphangiogenesis and counteract the negative effects of TGFβ on lymphangiogenesis by increasing the expression of VEGF-C. These findings provide new insights into tumor lymphangiogenesis and the various roles of TGFβ signaling in tumor regulation. Our results also suggest that SIX1/TGFβ might be a potential therapeutic target for preventing lymph node metastasis of tumor.